Although Staphylococcus aureus is not generally regarded as an intracellular pathogen, recent studies showed that this organism may be actively internalized by several types of cells. Working largely within an epithelial cell line, our laboratories partially characterized several steps of the cell invasion cycle employed by S. aureus. Based on our preliminary data,, and what is known about well-characterized intracellular pathogens, we propose a model that explains the interactions of. S. aureus with host cells. Three specific aims have been developed to extend our prior observations and focus our proposal on S. aureus entry, survival, and the host cell response to S. aureus internalization. Aim 1: Characterization of the S. aureus uptake mechanisms by epithelial cells. We will also determine if the requirement for fibronectin binding proteins is due to the fact that these proteins serve as the sole ligand for a host cell receptor or whether other molecules are required to bind to the receptor and induce signal transduction. We will also identify the host receptor (s) and partially characterize the signal transduction mechanisms Aim 2: Examination of S. aureus genes involved in intracellular survival. For this aim, we will use several molecular approaches to identify key genes and characterize their temporal regulation during the cell infection cycle cycle. We will also identify the putative membrane active staphylococcal product(s), which mediates escape from the endosome. Furthermore, we will carefully examine the potential role of the sp1 operon in the induction of apoptosis. Aim 3: Examination of the host cell response to intracellular S. aureus. Studies in this aim will build upon initial experiments demonstrating that internalized S. aureus leads to increased accumulation of various cytokine transcripts. To accomplish this, we will take advantage of recent advances in microarray technology to examine global host cells changes in gene expression.